- Email: [email protected]
Effect of aging and endurance training on tissue catecholamine response to strenuous exercise in Fischer 344 rats
Effect of Aging and Endurance Training on Tissue Catecholamine Strenuous Exercise in Fischer 344 Rats
Response to
Robert S. Mazzeo, Robert W. Colburn, and Steven M. Horvath The purpose of the present investigation was to determine the catecholamine response in various tissues to a bout of strenuous exercise in young, adult, and old Fischer 344 rats. Further, to study the effect of endurance training on this response, animals from each age group underwent ten weeks of treadmill running at 75% of their functional capacity. On completion
of the training
program,
all animals demonstrated
significant
increases
(P < 0.05)
in VO,max
and endurance
capacity. At rest or immediately after an acute bout of strenuous exercise, animals ware killed, and the heart, liver, kidney, and adrenals were removed for subsequent catecholamine analysis. Resting cardiac catecholamine levels declined significantly with age. In response to an acute exercise bout, epinephrine (El levels in the heart were greatly reduced with age averaging 141.8. 82.3, and 21.7 rig/g for the 8-, 15-. and 27-month-old untrained group, respectively. The 15- and 27-month-old trained animals demonstrated significantly higher E levels (33% and 91%) than controls. A similar trend was found for norepinephrine (NE) content in the heart in response to acute exercise, with a marked reduction occurring with advancing age (904.8, 580.1, and 400.8 rig/g heart for 8-. 15-, and 27-month-old untrained groups, respectively). Again, training induced a greater NE response in the older trained animals compared to age-matched controls. In contrast, adrenal catecholamine levels showed a tendency to increase with age. It was concluded that when challenged with strenuous physical stress, cardiac catecholamine content is markedly diminished with age. Further, ten weeks of endurance training can attenuate this functional decline. B 1986 by Grune & Stratton, Inc.
T
HE ABILITY
of an organism to respond to environmental stimuli declines with advancing age, thereby making it more difficult to physiologically adjust to stressful situations. More specifically, the ability to adjust to the stress of physical exercise, as measured by maximal levels of cardiac output,‘-3 heart rate,3T4myocardial contractility, and blood flow5-” is reduced with age. The two major regulatory pathways responsible for maintaining homeostasis under stressful conditions are the nervous and endocrine systems. Each has been reported to functionally diminish with age.‘2-‘4 Catecholamines play an integral role in adaptive processes as epinephrine (E) regulates a number of metabolic and physiologic functions and norepinephrine (NE) reflects the actions of the sympathetic nervous system. While resting levels of catecholamines in various tissues, such as the heart, the response of E and NE decline with advancing age, 13~‘5*‘6 to an intense bout of exercise, as well as with training, in older populations remains unclear. It was the purpose of this study to measure the catecholamine levels in various tissues in response to an acute maximal exercise bout in rats of 6, 15, and 27 months of age. Further, to determine the effect of physical conditioning on this response, animals from each age group were endurance trained for ten weeks.
From the institute of Environmental Stress, University of California, Santa Barbara. Calif Supported by a grant from the American Heart Association, California A@liate/Santa Barbara Chapter. Address reprint requests to Robert S. Mazzeo, PhD, Human Performance Laboratory, Box 356, University of Colorado, Boulder, CO 80309. Q 1986 by Grune & Stratton, Inc. 0026-0495/86/3507~00S$03.00/0
MATERIALS
AND METHODS
Animals
Female Fischer 344 rats, obtained through the National Institute of Aging (Charles River Breeding Laboratories, Wilmington, Mass), were received at the ages of 3,12, and 24 months. All animals were individually housed in climate-controlled quarters (25 OC, 12 hour light-dark photoperiod) and received Purina rodent chow and water ad libitum. Body weights for all animals were recorded weekly throughout the study with the weights being recorded at the same time of day. Training
Procedures
and Experimental
Protocol
All animals in each age group performed a maximal oxygen consumption (VO,max) test prior to training. We have previously reported on the VO,max procedures in these experimental animals.” The animals within a given age group were then pair-matched based on VO,max and assigned to either a sedentary or trained group. Training consisted of running 5 d/wk at an intensity of 75% VO,max for ten weeks. Animals initially ran for ten minutes up a 15% gradient at a speed dependent on age. Running time was progressively increased 5 min/d until animals were running continuously for 1 h/d. Thereafter, running speed was increased weekly to maintain training intensity while the duration was held constant at 1 h/d. Initial training speeds averaged 27.0,20.4, and 17.4 m/min for the young, middle-aged, and old animals, respectively. Sedentary control animals were run once a week for 5 minutes to familiarize them with treadmill running and handling. At the end of the ten-week training session, all animals were again tested for determination of VOrmax as well as endurance capacity. On days assigned for tissue catecholamine measurements (three to five days after the last training session), animals were killed either at rest or immediately at the point of fatigue during a graded exercise test. The progressive exercise test was administered with the animal initially running at 13.4 m/min on a 15% grade. The treadmill speed was increased 6.7 m/min every two minutes until the animal was unable to maintain pace with the treadmill. At the point of fatigue, the animal was immediately killed by decapitation.
Metabolism, Vol35, No 7 (July), 1966: pp 602-607
TISSUE CATECHOLAMINE
603
RESPONSE TO EXERCISE
Running times to fatigue averaged 11.2, 7.4, and 6.3 minutes in untrained age groups and, 18.7, 14.6, and 12.8 minutes for the trained animals aged 6, 15, and 27 months, respectively. The heart, liver, and kidney were then quickly removed, rinsed in ice-cold saline, blotted dry, weighed, and freeze-clamped in liquid nitrogen. In addition, the adrenal glands were also removed and all tissues were stored at - 80 OC for subsequent weighing and catecholamine analysis. The tissue sampling procedure totalled approximately 40 seconds from death of the animal. Ages at time of death were 6, 15, and 27 months for the respective groups.
control groups, as well as the difference across age, were determined by means of analysis of variance with post hoc Tukey analysis.
Tissue Analysis
Training EJect
All assays for catecholamine levels on a specific tissue were performed on the same day. Catecholamine levels were determined by means of high pressure liquid chromatography (HPLC) as modified by Hallman et al.‘* Sections of the tissue to be assayed were dissected free, weighed, and then homogenized in 2.6 mL of 0.1 N perchloric acid (PCA) at 5 “C. The homogenate was centrifuged at 1,000 g for three minutes. An internal standard was prepared by adding appropriate levels of DHBA (Dihydroxybenzylamine, Sigma, St Louis) in 50 rL of 0.1 N PCA to the supernate. After the addition of 100 PL of IO mmol/L sodium meta-bisulfite (NaHSO,, Fisher, San Francisco) to control catechole oxidation, the solution was pH adjusted to above 8.0 with 1.5mol/L tris buffer at pH 8.6 in 2% EDTA (Sigma, St Louis). Twenty-five milligrams of alumina (Woelm, ICN Pharmaceuticals), washed as described by Anton and Sayre,” were added followed by ten minutes of vigorous shaking. The alumina was then washed 4 x with 3 mL of distilled water with brief centrifugation between washes. The catecholamines were extracted with 200 rL of 0.1 N PCA for three minutes with shaking and final centrifugation at 12,000 g. Overall recoveries averaged more than 75%. Eighty microliters of eluant were then injected into the HPLC column (Cl8 reverse phase, 3 micron diameter, microsorb 80-200, Rainin, Emeryville, Calif), and eluted with mobile phase (14.15 g mono-chloroacetic, 4.67 g NaOH, 1.5 g Na,EDTA and 14 mg sodium octyl sulfate as an ion-pair reagent). The flow rate was 2.0 mL/min at 3,200 psi with 0.65 volts. The chromatogram was recorded on a RYT recorder (Bioanalytical Systems, Inc, West Lafayette Ind).
We have previously reported evidence of a training effect in these animals.” Anticipated increases in GO,max experienced from such a training program were found (1 I%, 18%, and 20% increases in q02max for the 6-, 15-, and 27 month-old animals, respectively). In addition, the trained animals across all age groups demonstrated a reduction in the respiratory exchange ratio (R) compared to the sedentary control groups for any given absolute or relative submaximal workload. Finally, the trained animals were able to achieve an increase in endurance times ranging from 350% to 800% of age-matched controls.
RESULTS
During the course of the experiment, four of the animals suffered fatalities (1 Un-12 mo, 2 Un-24 months, and 1 Tr-24 month). All the remaining animals were able to successfully complete the study. Body weights for all animals after the ten-week training program are shown in Table 1.
Heart and Adrenal Weights
The heart and adrenal weights are presented in Table 1. Heart weights increased significantly with age (P c 0.05). A difference in heart weight with training occurred only in the 27-month-old group, with the trained animals demonstrating significantly greater values when measured as both absolute and relative weights. Adrenal weights showed a tendency to increase with age. There were no significant differences between adrenal weights in any age group as a result of training when measured in absolute terms, however, when corrected for body weight, the 27-month-old trained animals demonstrated greater adrenal/body weight ratios. Catecholamine Response
Statistics
Resting cardiac catecholamine levels declined with age (Fig 1). E gradually decreased with age as the 27-month-old animals demonstrated significantly lower levels than the
Data are reported as means * SEM. Statistical differences in catecholamine content for a specific tissue between trained and
Table 1. Body, Heart, and Adrenal Weights BodVwt (9)
Heart Wt (me)
AdrenalWt lms)
Adrenal/BodyWt (mg/g)
6 Months Un (5)
176.7
+ 7.1
596 + 25
37.7
2 1.6
0.217
+ 0.026
Tr (5) 15 Months
172.6
f 4.7
594 k 27
39.0
+ 1.1
0.227
k 0.012
Un (5)
210.8
+ 6.0”
705 f
18*
42.8
f 3.2
0.181
+ 0.005”
Tr (5) 27 Months
212.4
+ 5.2*
698 + lo*
41.2
+ 1.9
0.178
r 0.009*
Un (6)
254.4
f 0.8t
792 + 20t
41.8
+ 1.9
0.165
f 0.007t
Tr (6)
229.8
+ 4.9t$
873 + 28t$
43.0
f 2.6
0.177
* o.oos*$
Values are means k SEM. Values in parentheses are the number of animals per group. *Significantly different from six-month-old animals (P < 0.05). tsignificantly different from 6-and
15-month-old animals P < 0.05).
SSignificantly difference between Un (untrained) and Tr (trained) animals (P -C 0.05).
604
MAZE0
+ Ex
160
1
A
content with increasing age (Fig 1A). Levels for E for the untrained groups averaged 141.8,62.3, and 21.7 rig/g heart for the 6-, 15, and 27-month-old animals, respectively. While the six-month-old untrained animals demonstrated significantly greater E levels (40%) when compared to the trained group, this finding was reversed in the 15 and 27-month-old groups as the trained animals had significantly greater E levels (33% and 91%) than controls. A similar trend was found for cardiac NE content (Fig 1B) with a pronounced decrease occurring in the response to strenuous exercise with age. Values averaged 904.6, 580.1, and 400.8 rig/g heart for the 6-, 15, and 27-month-old untrained groups, respectively. As with the E response, NE levels were higher in the 6-month-old control group compared to the trained animals (47%), but the trained older animals demonstrated a greater response (2% and 26%) when compared to the age-matched controls. When expressed in absolute terms, the NE content of the whole heart in response to exercise still demonstrated a decline with age in the untrained animals (540.2, 410.0, and 318.3 ng/heart: 6-, 15-, and 27-monthold, respectively). This finding was reversed in the trained groups as there was a tendency for total heart NE to be greater with increasing age (370.1, 413.7, and 443.7 ng/ heart: 6-, 15-, and 27-month-old, respectively). Catecholamine levels for the liver and kidney in response to strenuous exercise are presented in Table 2. While there was a trend for the catecholamine levels in these tissues to be lower with age, the only significant difference existed in the kidney between the 6-month-old and 27-month-old animals. In contrast to the response in the other tissues, the trend was for adrenal catecholamine content to be higher with age both at rest and in response to a maximal bout of exercise (Fig 2). Epinephrine levels averaged 483.8,485.0, and 538.1 rig/g adrenal for the 6-, 15-, and 27-month-old sedentary control animals, respectively. Adrenal epinephrine levels were significantly greater (P <: 0.05) in the trained animals for any given age increasing 15%, 44%, and 15% above the controls for the 6-, 15-, and 27-month-old animals, respectively. A similar response was witnessed for the NE levels as they demonstrated a tendancy to be greater with age averaging 74.6, 72.0, and 82.3 rig/g adrenal for the 6-, 15-, and 27-month-old control groups, respectively. Again, the NE levels were significantly higher for the trained groups averaging 83.6, 99.8, and 98.3 rig/g adrenal in the 6-, 15-, and 27-month-old animals.
120. Un t=: zk
Tr
Ex i’
1oo-
cl
ze 2 P
60.
w”P y
60.
Ex
f
40.
01 1200,
B Ex +
iooo* E-4 2:
800
$2 =Ul
600,
%k $5 2
Re Ex t*
400, 200,
0 15
6
27
Age (months) Heart epinephrine (A) and norepinephrine (6) levels Fig I. (mean + SEMI at rest (Re) and after a bout of strenuous exercise (Ex) in trained and untrained rats ages 6, 16, and 27 months. N = 6.6; 46; and 6.6 for untrained and trained rats of the 6.16, and 27 month groups, respectively. TSignificantly different from rest (P i 0.06). *Significant difference between trained and untrained animals. “Significantly different from six-month-old animals. %ignificantly different from 8 and 16-month-old animals.
younger groups. Resting NE levels declined more dramatically with significant differences found between each age group. In response to a maximal bout of exercise, cardiac catecholamine levels differed greatly between both trained and untrained groups as well as between animals of different ages. There was a clear and significant reduction in heart E
Table 2.
NE Content
in the Liver and Kidney at Rest and in Response to a Strenuous Liver
Rest
97.1
+ 6.8 (10)
68.6
+ 4.2.
Bout of Exercise Kidney
27 Months
16 Months
6 Months
ET AL
(10)
46.4
f 3.8t
16 Months
6 Months
(12)
236.7
f 20.0(10)
200.1
27 Months
f 16.7’
(10) (5)
160.4
+ 16.3t
(12)
241.6
f 20.4t
(6)
262.6
+ 13.6t
(6)
Exercise Un
96.2
f 11.6 (6)
86.0
f 13.8 (6)
84.1
+ 12.1 (6)
374.8
+ 43.0 (6)
293.0
+ 30.4’
Tr
96.9
f 9.6 (6)
90.6
f
66.5
+ 19.8’
342.4
f
381.6
i 27.3 (6)
Values are means
f
18.3 (6)
(8)
10.7 (6)
SEM.
Values in parentheses are the number of animals par group. All units are expressed as nQ/g tissue. Resting values for Un and Tr animals were pooled. *Significantly different from six-month-old animals (P < 0.05). tsignificantly different from 6- and 15-month-old animals (P < 0.06).
TISSUE CATECHOLAMINE
605
RESPONSE TO EXERCISE
RI?
Re
EX f
Re
Fx
Ex
6
Age (months) Fig 2. Adrenal epinephrine (A) and norepinephrine (6) levels (mean + SEMJ at rest (Re) and after a bout of strenuous exercise (Exl in trained and untrained rats ages 6. 16. and 27 months. N = 6.5: 4.5; and 5.6 for the untrained and trained animals in the 6, 15-. and 27-month-old groups, respactivefy. tSignificantly different from rest (P -C 0.06). *Significant difference between the trained and untrained animals. “Significantly different from sixmonth-old animals.
DISCUSSION
A number of investigators have reported the resting levels of catecholamines to be reduced with age in the heart of the rat. ‘3~‘5*16 While this response was more pronounced in the case of NE than E, both declined significantly with age (Fig 1). A similar response occurs as a consequence of strenuous exercise as well. There is a clear and significant decline in the cardiac catecholamine levels with increasing age. Compared to rest, heart E levels rose markedly during exercise. As the major source of this E under such conditions is from adrenal release into the blood, E presence in the heart reflects its role as a hormone. There was a sharp reduction with age in the untrained animals’ E response to exercise. While this response was somewhat attenuated with training, a significant decline still persisted with age. The six-month-old trained animals demonstrated significantly lower cardiac E levels compared to the untrained group. This finding is in agreement with previous studies on rats and humans showing reduced plasma catecholamine levels with training in response to a given absolute or relative workload.20-22 An increased sensitivity of the &adrenergic receptors has been suggested as one possible mechanism responsible for this training effect. This finding, however, is reversed in the 15 and 27-month-old animals as the trained
groups recorded significantly higher cardiac E values in response to strenuous exercise when compared to untrained rats. This suggests that some age-related mechanism(s) has intervened and has effected the training response of catecholamine metabolism. Resting cardiac NE content decreased with age. However, in response to exercise, NE levels did not increase as drastically as seen with E. The major presence of NE found in the heart reflects its role as a sympathetic neurotransmitter. Consequently, during the stimulus of strenuous exericse, sympathetic activity as well as NE synthesis is increased.2”5 However, depending on the ability of the vesicles for reuptake of the released NE, tissue levels can increase, decrease, or be maintained. As with E, NE levels in the untrained animals declined sharply with age during exercise. However, this decline was greatly attenuated in the trained groups with only the 27-month-old group demonstrating statistically lower values when compared to younger animals. The six-month-old trained animals had lower NE levels in response to exercise compared to the control animals, but as with E, this finding was reversed in the older animals. Again, this suggest some age-related alteration in the training adaptation to catecholamine metabolism; however, in the case of NE it is primarily in its capacity as a neurotransmitter while the E response is mainly hormonal. However, the major source of E is from the adrenal medulla, and this tissue is under direct sympathetic control. Consequently, it generally reflects the actions of the sympathetic nervous system. There was a trend for adrenal catecholamines to be higher with age both at rest and in response to strenuous exercise. Compared to rest, levels of catecholamines decreased during exercise in untrained animals across all ages as these hormones were released into the blood to help adjust to the metabolic demands imposed by the exercise bout. However, the trained animals were able to maintain adrenai levels similar to rest. The present study has examined the tissue catecholamine response during strenuous exercise in young, middle-aged, and old rats. While no other reports exist regarding tissue catecholamine response to exercise in older rats, studies on the heart catecholamines in younger rats indicate there is an increase in sympathetic activity.23*25V26 Several investigators have found exercise to produce transient increases in the catecholamine levels in the heart.23~25Further, using (Ymethyl-tyrosine and “C-tyrosine, Gordon et a124have determined that the increased sympathetic stimulus associated with acute exercise induces an increase rate of synthesis of NE and E during the course of exercise to keep pace with the elevated sympathetic drive. This rate increase in the synthesis of catecholamines was shown to occur in the heart, adrenal, and brain of the rat. Thus, any impairment on the capacity for synthesis, release, or re-uptake of catecholamines would result not only in lower concentrations of catecholamines in the active tissues, but as a diminished functional response as well. Evidence for such an impaired catecholamine response in the older animals is suggested by diminished cardiac catecholamine levels. While there was a trend for the tissue catecholamine levels to decline with age in the liver and kidney, the only significant difference
MAZE0
occurred between the 6- and 27-month-old animals in the kidney. This may be related to the degree of recruitment and distribution of blood to these tissues during exercise. It is evident that during the course of exercise, endurance training reduces the amount of circulating catecholamines in response to a given workload.*“** It has been suggested that this observation is a result of increased sensitivity to catecholamines by receptors. Winder et al*’ demonstrated in humans that the time course for this training-induced blood catecholamine response is approximately three weeks. Therefore, the ten-week training regimen employed in the present study was of sufficient duration to induce such a response. The finding in the present study that in response to strenuous exercise the catecholamine levels in the hearts of the young animals were greater in the untrained compared to the trained group provides further evidence for this hypersensitivity effect. However, in the 15 and 27-month-old animals the opposite response was observed in which the trained animals had higher levels of cardiac catecholamines than did the untrained group. This suggests that when compared to the young animals some other factor(s) has intervened, which has limited the older untrained animals’ ability for catecholamine synthesis, release, or re-uptake during the exercise stimulus and that training has offset this decline to a certain extent. Regardless of the training state, the 15 and 27-month-old animals demonstrated a significantly lower catecholamine response compared to the younger animals. Ten weeks of endurance training can significantly retard this age-related functional decline. The sympathetic nervous system, as well as the endocrine system, plays a major role in enabling the organism to adapt and maintain homeostasis in response to physical stress. The finding that the 27-monthold trained animals demonstrated significantly greater NE and E levels in the heart after strenuous exercise suggests that these animals are better adapted to cope with the demands of physical stress than their age-matched control group. While increased levels of cardiac catecholamines do not necessarily imply increased performance, the catecholamines have a pronounced effect on myocardial metabolism and function including enhanced glucose metabolism, increased rate, contractility, and cardiac output. Lakatta et al*’ has shown that the myocardial function, as measured by maximal rate of tension development (dT/dt), active tension, and contraction duration, to be greatly diminished in the aged heart in response to catecholamine administration. However, regardless of age, the inotropic response of the myocardium was greater with increasing catecholamine concentrations. This, in part, could explain the reductions in maximal stroke volume, cardiac output, heart rate, and functional capacity observed with aging. Further, Wyatt et al29 demonstrated that endurance training significantly improves the catecholamine-induced enhancement of myocardial contractile function and adenylate cyclase activity in the cat. Therefore, it would appear that the 27-monthtrained animals have a functional advantage in their ability to respond to the demands of intense physical exercise compared to their age-matched controls. The training stimulus improves the animals’ ability to respond to physical stress
ET AL
both in terms of the absolute catecholamine response and in the myocardium’s ability to respond to the catecholamines presented to it. This suggests that although the ageassociated functional decline may be related to the “aging process ” itself, other factors such as disuse and sedentary lifestyles can promote such changes. The reader should be aware of two factors that could possibly influence catecholamine levels of the present study. First, while all the animals ran at the same relative intensity, the running times to fatigue during the graded exercise test varied with age and training status. This could potentially affect the plasma catecholamine content. However, two lines of evidence suggest that this would not significantly alter our interpretation of the data. The six-month-old trained animals ran significantly longer than did the untrained animals of the same age (18.7 v 11.2 minutes), yet the trained animals demonstrated significantly lower NE and E levels in the heart. Further, this trend was reversed in the 15 and 27-month-old animals as the trained animals demonstrated higher catecholamine values when compared to their agematched controls. This reversal in response clearly indicates that some age-related factor(s) has intervened, which has effected the training response of catecholamine metabolism. In addition, while there were very small differences in run times to fatigue between the IS- and 27-month-old animals (Tr = 14.6 v 12.8 : Un = 7.4 v 6.3), there were significant differences in catecholamine content with age in response to exercise (Fig I). The method of death in the present study was decapitation. This represents a strong stimulus to the animal and is known to markedly elevate plasma catecholamines. This, in conjunction with the stimulus of strenuous exercise, could explain the relatively high levels of E observed in the heart. The combination of several factors including (1) a small amount of trapped blood remaining in the heart, (2) uptake of E into cardiac sympathetic terminals,3”32 and (3) increased synthesis of E by chromaffin cells within the heart33*34 most likely contribute to the relatively high E values observed in the heart after strenuous exercise. Regardless, in subsequent studies in our laboratory examining NE turnover in various tissues with age, an anesthetic was used to kill the animals. While cardiac catecholamine levels were slightly depressed compared to the results of the present study (most noticeable at rest but statistically insignificant), a similar response was observed as heart catecholamine content, as well as NE turnover rates, were significantly depressed with age. Based on previous literature regarding changes in the ability for catecholamine synthesis with advancing age, together with the results of the present study, it would suggest that the mechanism responsible for the diminished catecholamine levels in the aged rats during strenuous exercise is, in part, a result of a reduced capacity for catecholamine synthesis. However, data on tissue catecholamines alone is insufficient to provide conclusive evidence relating to sympathetic activity. Differences in catecholamine concentrations may reflect changes in the ability for synthesis and release, volume distribution, and clearance as determined by local metabolism and re-uptake into nerve terminals. While a
TISSUE
CATECHOLAMINE
RESPONSE
607
TO EXERCISE
number of recent reports have provided evidence demonstrating that tissue and circulating norepinephrine levels are a reliable index of underlying sympathetic activity,3C37 properly designed NE turnover studies need to be conducted to determine the exact mechanism responsible. However, the
results of the present study clearly indicate an age-related decline in cardiac catecholamine levels in response to strenuous exercise and that ten weeks of endurance training can significantly attenuate this response.
REFERENCES
1. Julius S, Amery A, Whitlock LS, et al: Influence of age on the hemodynamic response to exercise. Circulation 36:222-230, 1967 2. Lakatta EC: Age-related alterations in the cardiovascular responses to adrenergic mediated stress. Fed Proc 39:3173-3177, 1980 3. Shephard RJ: Cardiovascular limitations in the aged, in Smith EL, Serfass RC (eds): Exercise and Aging: The Scientific Basis. Hillsider, NJ, Enslow 1981, pp 19-29 4. Astrand I, Astrand PO, Rodahl K: Maximal heart rate during work in older men. J Appl Physiol 14:562-566, 1959 5. Alpert NR, Gale HH, Taylor N: The effect of age on contractile protein ATPase activity and the velocity of shortening, in: Kavelar K, Tanz RD, and Roberts J (eds): Factors Influencing Myocardial Contractility. Orlando, Fla, Academic, 1967, pp 127133 6. Capasso JM, Malhotra A, Remily RM, et al: Effects of age on mechanical and electrical performance of rat myocardium. Am J Physiol 245:H72-H81, 1983 7. Gerstenblith G, Lakatta EG, Weisfeldt ML: Age changes in myocardial function and exercise response. Prog Cardiovasc Dis 19:1-21, 1976 8. Guarnieri T, Filburn CR, Zitnik G, et al: Contractile and biochemical correlates of B-adrenergic stimulation of the aged heart. Am J Physiol239:H501-H508.1980 9. Heller LJ, Whitehorn WV: Age-associated alterations in myocardial contractile properties. Am J Physiol222:1613-1619, 1972 10. Starnes JW, Beyer RE, Edington DW: Myocardial adaptations to endurance exercise in aged rats. Am J Physiol 245:560-566, 1983 1.1. Weisfeldt ML, Wright JR, Shreiner DP, et al: Coronary flow and oxygen extraction in the perfused heart of senescent male rats. J Appl Physiol 30:4449, 197 1 12. Conway J, Wheeler R, Sannerstedt R: Sympathetic nervous activity during exercise in relation to age. Cardiovasc Res 5:577581, 1971 13. Frolkis VV, Bezrukov VV, Bogatskaya LN, et al: NS Catecholamines in the metabolism and functions regulation in aging. Gerontologia 16: 129-l 40, 1970 14. Timiras PS: Developmental Physiology and Aging. New York, Macmillan, 1972, pp 542-563 15. Gey KF, Burkard WP, Pletscher A: Variation of the norepinephrine metabolism of the rat heart with age. Gerontologia 1 l:l11, 1965 16. Roberts J, Goldberg PB: Changes in basic cardiovascular activities during the lifetime of the rat. Exp Aging Res 2:487-517, 1976 17. Mazzeo RS, Brooks GA, Horvath SM: Effects of age on the metabolic responses to endurance training in rats. J Appl Physiol 57:1369-1374,1984 18. Hallman H, Farnebo L, Hamberger B, et al: A sensitive method for the determination of plasma catecholamines using liquid chromatography with electrical detection. Life Sci 23:1049-1052, 1978
19. Anton AH, Sayre DF: A study of the factors affecting the aluminum oxide-trihydroxyindole procedure for the analysis of catecholamines. J Pharmacol Exp Ther 138:360-375, 1962 20. Galbo H, Richter EA, Hoist JJ: Diminished hormonal responses to exercise in trained rats. J Appl Physiol 43:953-958, 1977 21. Winder WW, Hickson RC, Hagberg JM, et al: Traininginduced changes in hormonal and metabolic responses to submaximal exercise. J Appl Physiol46:766-77 1, 1979 22. Winder WW, Beattie MA, Holman RT: Endurance training attenuates stress hormone responses to exercise in fasted rats. Am J Physiol243:R179-R184,1982 23. De Schryver C, Strythagen J, Becsei I, et al: Effect of training on heart and skeletal muscle catecholamine concentration in rats. Am J Physiol217:1589-1592, 1969 24. Gordon R, Spector S, Sjoerdsma A, et al: Increased synthesis of norepinephrine and epinephrine in the intact rat during exercise and exposure to cold. J Pharmacol Exp Ther 153:440-447,1966 25. Nikkila EA, Torsti P, Penttila 0: Effects of fasting, exercise and reserpine on catecholamine content and lipoprotein lipase activity of rat heart and adipose tissues. Life Sci 4:27-35, 1965 26. Leon AS, Horst WD, Spit? N, et al: Heart norepinephrine levels after exercise training in the rat. Chest 67:341-343, 1975 27. Winder WW, Hagberg JM, Hickson RC, et al: Time course of sympathoadrenal adaptation to endurance exercise training in man. J Appl Physiol45:37&374, 1978 28. Lakatta EG, Gerstenblith GS, Angel1 CS, et al: Diminished inotropic response of aged myocardium to catecholamines. Circ Res 361262-269, 1975 29. Wyatt HL, Chuck L, Rabinowitz B, et al: Enhanced cardiac response to catecholamines in physically trained cats. Am J Physiol 234:H608-H613,1978 30. Rose11S, Axelrod J, Kopin IJ: Release of tritated epinephrine following sympathetic nerve stimulation. Nature 201:301, 1964 3 1. Stromblad BCR, Nickerson M: Accumulation of epinephrine and norepinephrine by some rat tissues. J Pharmacol Exp Ther 134:154-159, 1961 32. Iversen LL: The uptake of catechol amines at high perfusion concentrations in the rat isolated heart: A novel catechol amine uptake process. Br J Pharmacol25:18-33, 1965 33. Glassman PM, Angelakos ET, McNary WF: Catecholaminecontaining fractions of dog heart homogenates. Life Sci 4:17271734, 1965 34. Potter LT, Axelrod J: Subcellular localization of catecholamines in tissues of the rat. J Pharmacol Exp Ther 142:291-298, 1963 35. Christensen NJ: Catecholamines and sympathetic nervous activity in the elderly. Acta Med Stand 676:52-63, 1983 (suppl) 36. Rowe JW, Troen BR: Sympathetic nervous system and aging in man. Endocr Rev 1:167-179, 1980 37. Wallin BG, Sundolf G, Eriksson B, et al: Plasma noradrenaline correlates to sympathetic muscle nerve activity in normotensive man. Acta Physiol Stand 111:69-73, 1981
Response to
Robert S. Mazzeo, Robert W. Colburn, and Steven M. Horvath The purpose of the present investigation was to determine the catecholamine response in various tissues to a bout of strenuous exercise in young, adult, and old Fischer 344 rats. Further, to study the effect of endurance training on this response, animals from each age group underwent ten weeks of treadmill running at 75% of their functional capacity. On completion
of the training
program,
all animals demonstrated
significant
increases
(P < 0.05)
in VO,max
and endurance
capacity. At rest or immediately after an acute bout of strenuous exercise, animals ware killed, and the heart, liver, kidney, and adrenals were removed for subsequent catecholamine analysis. Resting cardiac catecholamine levels declined significantly with age. In response to an acute exercise bout, epinephrine (El levels in the heart were greatly reduced with age averaging 141.8. 82.3, and 21.7 rig/g for the 8-, 15-. and 27-month-old untrained group, respectively. The 15- and 27-month-old trained animals demonstrated significantly higher E levels (33% and 91%) than controls. A similar trend was found for norepinephrine (NE) content in the heart in response to acute exercise, with a marked reduction occurring with advancing age (904.8, 580.1, and 400.8 rig/g heart for 8-. 15-, and 27-month-old untrained groups, respectively). Again, training induced a greater NE response in the older trained animals compared to age-matched controls. In contrast, adrenal catecholamine levels showed a tendency to increase with age. It was concluded that when challenged with strenuous physical stress, cardiac catecholamine content is markedly diminished with age. Further, ten weeks of endurance training can attenuate this functional decline. B 1986 by Grune & Stratton, Inc.
T
HE ABILITY
of an organism to respond to environmental stimuli declines with advancing age, thereby making it more difficult to physiologically adjust to stressful situations. More specifically, the ability to adjust to the stress of physical exercise, as measured by maximal levels of cardiac output,‘-3 heart rate,3T4myocardial contractility, and blood flow5-” is reduced with age. The two major regulatory pathways responsible for maintaining homeostasis under stressful conditions are the nervous and endocrine systems. Each has been reported to functionally diminish with age.‘2-‘4 Catecholamines play an integral role in adaptive processes as epinephrine (E) regulates a number of metabolic and physiologic functions and norepinephrine (NE) reflects the actions of the sympathetic nervous system. While resting levels of catecholamines in various tissues, such as the heart, the response of E and NE decline with advancing age, 13~‘5*‘6 to an intense bout of exercise, as well as with training, in older populations remains unclear. It was the purpose of this study to measure the catecholamine levels in various tissues in response to an acute maximal exercise bout in rats of 6, 15, and 27 months of age. Further, to determine the effect of physical conditioning on this response, animals from each age group were endurance trained for ten weeks.
From the institute of Environmental Stress, University of California, Santa Barbara. Calif Supported by a grant from the American Heart Association, California A@liate/Santa Barbara Chapter. Address reprint requests to Robert S. Mazzeo, PhD, Human Performance Laboratory, Box 356, University of Colorado, Boulder, CO 80309. Q 1986 by Grune & Stratton, Inc. 0026-0495/86/3507~00S$03.00/0
MATERIALS
AND METHODS
Animals
Female Fischer 344 rats, obtained through the National Institute of Aging (Charles River Breeding Laboratories, Wilmington, Mass), were received at the ages of 3,12, and 24 months. All animals were individually housed in climate-controlled quarters (25 OC, 12 hour light-dark photoperiod) and received Purina rodent chow and water ad libitum. Body weights for all animals were recorded weekly throughout the study with the weights being recorded at the same time of day. Training
Procedures
and Experimental
Protocol
All animals in each age group performed a maximal oxygen consumption (VO,max) test prior to training. We have previously reported on the VO,max procedures in these experimental animals.” The animals within a given age group were then pair-matched based on VO,max and assigned to either a sedentary or trained group. Training consisted of running 5 d/wk at an intensity of 75% VO,max for ten weeks. Animals initially ran for ten minutes up a 15% gradient at a speed dependent on age. Running time was progressively increased 5 min/d until animals were running continuously for 1 h/d. Thereafter, running speed was increased weekly to maintain training intensity while the duration was held constant at 1 h/d. Initial training speeds averaged 27.0,20.4, and 17.4 m/min for the young, middle-aged, and old animals, respectively. Sedentary control animals were run once a week for 5 minutes to familiarize them with treadmill running and handling. At the end of the ten-week training session, all animals were again tested for determination of VOrmax as well as endurance capacity. On days assigned for tissue catecholamine measurements (three to five days after the last training session), animals were killed either at rest or immediately at the point of fatigue during a graded exercise test. The progressive exercise test was administered with the animal initially running at 13.4 m/min on a 15% grade. The treadmill speed was increased 6.7 m/min every two minutes until the animal was unable to maintain pace with the treadmill. At the point of fatigue, the animal was immediately killed by decapitation.
Metabolism, Vol35, No 7 (July), 1966: pp 602-607
TISSUE CATECHOLAMINE
603
RESPONSE TO EXERCISE
Running times to fatigue averaged 11.2, 7.4, and 6.3 minutes in untrained age groups and, 18.7, 14.6, and 12.8 minutes for the trained animals aged 6, 15, and 27 months, respectively. The heart, liver, and kidney were then quickly removed, rinsed in ice-cold saline, blotted dry, weighed, and freeze-clamped in liquid nitrogen. In addition, the adrenal glands were also removed and all tissues were stored at - 80 OC for subsequent weighing and catecholamine analysis. The tissue sampling procedure totalled approximately 40 seconds from death of the animal. Ages at time of death were 6, 15, and 27 months for the respective groups.
control groups, as well as the difference across age, were determined by means of analysis of variance with post hoc Tukey analysis.
Tissue Analysis
Training EJect
All assays for catecholamine levels on a specific tissue were performed on the same day. Catecholamine levels were determined by means of high pressure liquid chromatography (HPLC) as modified by Hallman et al.‘* Sections of the tissue to be assayed were dissected free, weighed, and then homogenized in 2.6 mL of 0.1 N perchloric acid (PCA) at 5 “C. The homogenate was centrifuged at 1,000 g for three minutes. An internal standard was prepared by adding appropriate levels of DHBA (Dihydroxybenzylamine, Sigma, St Louis) in 50 rL of 0.1 N PCA to the supernate. After the addition of 100 PL of IO mmol/L sodium meta-bisulfite (NaHSO,, Fisher, San Francisco) to control catechole oxidation, the solution was pH adjusted to above 8.0 with 1.5mol/L tris buffer at pH 8.6 in 2% EDTA (Sigma, St Louis). Twenty-five milligrams of alumina (Woelm, ICN Pharmaceuticals), washed as described by Anton and Sayre,” were added followed by ten minutes of vigorous shaking. The alumina was then washed 4 x with 3 mL of distilled water with brief centrifugation between washes. The catecholamines were extracted with 200 rL of 0.1 N PCA for three minutes with shaking and final centrifugation at 12,000 g. Overall recoveries averaged more than 75%. Eighty microliters of eluant were then injected into the HPLC column (Cl8 reverse phase, 3 micron diameter, microsorb 80-200, Rainin, Emeryville, Calif), and eluted with mobile phase (14.15 g mono-chloroacetic, 4.67 g NaOH, 1.5 g Na,EDTA and 14 mg sodium octyl sulfate as an ion-pair reagent). The flow rate was 2.0 mL/min at 3,200 psi with 0.65 volts. The chromatogram was recorded on a RYT recorder (Bioanalytical Systems, Inc, West Lafayette Ind).
We have previously reported evidence of a training effect in these animals.” Anticipated increases in GO,max experienced from such a training program were found (1 I%, 18%, and 20% increases in q02max for the 6-, 15-, and 27 month-old animals, respectively). In addition, the trained animals across all age groups demonstrated a reduction in the respiratory exchange ratio (R) compared to the sedentary control groups for any given absolute or relative submaximal workload. Finally, the trained animals were able to achieve an increase in endurance times ranging from 350% to 800% of age-matched controls.
RESULTS
During the course of the experiment, four of the animals suffered fatalities (1 Un-12 mo, 2 Un-24 months, and 1 Tr-24 month). All the remaining animals were able to successfully complete the study. Body weights for all animals after the ten-week training program are shown in Table 1.
Heart and Adrenal Weights
The heart and adrenal weights are presented in Table 1. Heart weights increased significantly with age (P c 0.05). A difference in heart weight with training occurred only in the 27-month-old group, with the trained animals demonstrating significantly greater values when measured as both absolute and relative weights. Adrenal weights showed a tendency to increase with age. There were no significant differences between adrenal weights in any age group as a result of training when measured in absolute terms, however, when corrected for body weight, the 27-month-old trained animals demonstrated greater adrenal/body weight ratios. Catecholamine Response
Statistics
Resting cardiac catecholamine levels declined with age (Fig 1). E gradually decreased with age as the 27-month-old animals demonstrated significantly lower levels than the
Data are reported as means * SEM. Statistical differences in catecholamine content for a specific tissue between trained and
Table 1. Body, Heart, and Adrenal Weights BodVwt (9)
Heart Wt (me)
AdrenalWt lms)
Adrenal/BodyWt (mg/g)
6 Months Un (5)
176.7
+ 7.1
596 + 25
37.7
2 1.6
0.217
+ 0.026
Tr (5) 15 Months
172.6
f 4.7
594 k 27
39.0
+ 1.1
0.227
k 0.012
Un (5)
210.8
+ 6.0”
705 f
18*
42.8
f 3.2
0.181
+ 0.005”
Tr (5) 27 Months
212.4
+ 5.2*
698 + lo*
41.2
+ 1.9
0.178
r 0.009*
Un (6)
254.4
f 0.8t
792 + 20t
41.8
+ 1.9
0.165
f 0.007t
Tr (6)
229.8
+ 4.9t$
873 + 28t$
43.0
f 2.6
0.177
* o.oos*$
Values are means k SEM. Values in parentheses are the number of animals per group. *Significantly different from six-month-old animals (P < 0.05). tsignificantly different from 6-and
15-month-old animals P < 0.05).
SSignificantly difference between Un (untrained) and Tr (trained) animals (P -C 0.05).
604
MAZE0
+ Ex
160
1
A
content with increasing age (Fig 1A). Levels for E for the untrained groups averaged 141.8,62.3, and 21.7 rig/g heart for the 6-, 15, and 27-month-old animals, respectively. While the six-month-old untrained animals demonstrated significantly greater E levels (40%) when compared to the trained group, this finding was reversed in the 15 and 27-month-old groups as the trained animals had significantly greater E levels (33% and 91%) than controls. A similar trend was found for cardiac NE content (Fig 1B) with a pronounced decrease occurring in the response to strenuous exercise with age. Values averaged 904.6, 580.1, and 400.8 rig/g heart for the 6-, 15, and 27-month-old untrained groups, respectively. As with the E response, NE levels were higher in the 6-month-old control group compared to the trained animals (47%), but the trained older animals demonstrated a greater response (2% and 26%) when compared to the age-matched controls. When expressed in absolute terms, the NE content of the whole heart in response to exercise still demonstrated a decline with age in the untrained animals (540.2, 410.0, and 318.3 ng/heart: 6-, 15-, and 27-monthold, respectively). This finding was reversed in the trained groups as there was a tendency for total heart NE to be greater with increasing age (370.1, 413.7, and 443.7 ng/ heart: 6-, 15-, and 27-month-old, respectively). Catecholamine levels for the liver and kidney in response to strenuous exercise are presented in Table 2. While there was a trend for the catecholamine levels in these tissues to be lower with age, the only significant difference existed in the kidney between the 6-month-old and 27-month-old animals. In contrast to the response in the other tissues, the trend was for adrenal catecholamine content to be higher with age both at rest and in response to a maximal bout of exercise (Fig 2). Epinephrine levels averaged 483.8,485.0, and 538.1 rig/g adrenal for the 6-, 15-, and 27-month-old sedentary control animals, respectively. Adrenal epinephrine levels were significantly greater (P <: 0.05) in the trained animals for any given age increasing 15%, 44%, and 15% above the controls for the 6-, 15-, and 27-month-old animals, respectively. A similar response was witnessed for the NE levels as they demonstrated a tendancy to be greater with age averaging 74.6, 72.0, and 82.3 rig/g adrenal for the 6-, 15-, and 27-month-old control groups, respectively. Again, the NE levels were significantly higher for the trained groups averaging 83.6, 99.8, and 98.3 rig/g adrenal in the 6-, 15-, and 27-month-old animals.
120. Un t=: zk
Tr
Ex i’
1oo-
cl
ze 2 P
60.
w”P y
60.
Ex
f
40.
01 1200,
B Ex +
iooo* E-4 2:
800
$2 =Ul
600,
%k $5 2
Re Ex t*
400, 200,
0 15
6
27
Age (months) Heart epinephrine (A) and norepinephrine (6) levels Fig I. (mean + SEMI at rest (Re) and after a bout of strenuous exercise (Ex) in trained and untrained rats ages 6, 16, and 27 months. N = 6.6; 46; and 6.6 for untrained and trained rats of the 6.16, and 27 month groups, respectively. TSignificantly different from rest (P i 0.06). *Significant difference between trained and untrained animals. “Significantly different from six-month-old animals. %ignificantly different from 8 and 16-month-old animals.
younger groups. Resting NE levels declined more dramatically with significant differences found between each age group. In response to a maximal bout of exercise, cardiac catecholamine levels differed greatly between both trained and untrained groups as well as between animals of different ages. There was a clear and significant reduction in heart E
Table 2.
NE Content
in the Liver and Kidney at Rest and in Response to a Strenuous Liver
Rest
97.1
+ 6.8 (10)
68.6
+ 4.2.
Bout of Exercise Kidney
27 Months
16 Months
6 Months
ET AL
(10)
46.4
f 3.8t
16 Months
6 Months
(12)
236.7
f 20.0(10)
200.1
27 Months
f 16.7’
(10) (5)
160.4
+ 16.3t
(12)
241.6
f 20.4t
(6)
262.6
+ 13.6t
(6)
Exercise Un
96.2
f 11.6 (6)
86.0
f 13.8 (6)
84.1
+ 12.1 (6)
374.8
+ 43.0 (6)
293.0
+ 30.4’
Tr
96.9
f 9.6 (6)
90.6
f
66.5
+ 19.8’
342.4
f
381.6
i 27.3 (6)
Values are means
f
18.3 (6)
(8)
10.7 (6)
SEM.
Values in parentheses are the number of animals par group. All units are expressed as nQ/g tissue. Resting values for Un and Tr animals were pooled. *Significantly different from six-month-old animals (P < 0.05). tsignificantly different from 6- and 15-month-old animals (P < 0.06).
TISSUE CATECHOLAMINE
605
RESPONSE TO EXERCISE
RI?
Re
EX f
Re
Fx
Ex
6
Age (months) Fig 2. Adrenal epinephrine (A) and norepinephrine (6) levels (mean + SEMJ at rest (Re) and after a bout of strenuous exercise (Exl in trained and untrained rats ages 6. 16. and 27 months. N = 6.5: 4.5; and 5.6 for the untrained and trained animals in the 6, 15-. and 27-month-old groups, respactivefy. tSignificantly different from rest (P -C 0.06). *Significant difference between the trained and untrained animals. “Significantly different from sixmonth-old animals.
DISCUSSION
A number of investigators have reported the resting levels of catecholamines to be reduced with age in the heart of the rat. ‘3~‘5*16 While this response was more pronounced in the case of NE than E, both declined significantly with age (Fig 1). A similar response occurs as a consequence of strenuous exercise as well. There is a clear and significant decline in the cardiac catecholamine levels with increasing age. Compared to rest, heart E levels rose markedly during exercise. As the major source of this E under such conditions is from adrenal release into the blood, E presence in the heart reflects its role as a hormone. There was a sharp reduction with age in the untrained animals’ E response to exercise. While this response was somewhat attenuated with training, a significant decline still persisted with age. The six-month-old trained animals demonstrated significantly lower cardiac E levels compared to the untrained group. This finding is in agreement with previous studies on rats and humans showing reduced plasma catecholamine levels with training in response to a given absolute or relative workload.20-22 An increased sensitivity of the &adrenergic receptors has been suggested as one possible mechanism responsible for this training effect. This finding, however, is reversed in the 15 and 27-month-old animals as the trained
groups recorded significantly higher cardiac E values in response to strenuous exercise when compared to untrained rats. This suggests that some age-related mechanism(s) has intervened and has effected the training response of catecholamine metabolism. Resting cardiac NE content decreased with age. However, in response to exercise, NE levels did not increase as drastically as seen with E. The major presence of NE found in the heart reflects its role as a sympathetic neurotransmitter. Consequently, during the stimulus of strenuous exericse, sympathetic activity as well as NE synthesis is increased.2”5 However, depending on the ability of the vesicles for reuptake of the released NE, tissue levels can increase, decrease, or be maintained. As with E, NE levels in the untrained animals declined sharply with age during exercise. However, this decline was greatly attenuated in the trained groups with only the 27-month-old group demonstrating statistically lower values when compared to younger animals. The six-month-old trained animals had lower NE levels in response to exercise compared to the control animals, but as with E, this finding was reversed in the older animals. Again, this suggest some age-related alteration in the training adaptation to catecholamine metabolism; however, in the case of NE it is primarily in its capacity as a neurotransmitter while the E response is mainly hormonal. However, the major source of E is from the adrenal medulla, and this tissue is under direct sympathetic control. Consequently, it generally reflects the actions of the sympathetic nervous system. There was a trend for adrenal catecholamines to be higher with age both at rest and in response to strenuous exercise. Compared to rest, levels of catecholamines decreased during exercise in untrained animals across all ages as these hormones were released into the blood to help adjust to the metabolic demands imposed by the exercise bout. However, the trained animals were able to maintain adrenai levels similar to rest. The present study has examined the tissue catecholamine response during strenuous exercise in young, middle-aged, and old rats. While no other reports exist regarding tissue catecholamine response to exercise in older rats, studies on the heart catecholamines in younger rats indicate there is an increase in sympathetic activity.23*25V26 Several investigators have found exercise to produce transient increases in the catecholamine levels in the heart.23~25Further, using (Ymethyl-tyrosine and “C-tyrosine, Gordon et a124have determined that the increased sympathetic stimulus associated with acute exercise induces an increase rate of synthesis of NE and E during the course of exercise to keep pace with the elevated sympathetic drive. This rate increase in the synthesis of catecholamines was shown to occur in the heart, adrenal, and brain of the rat. Thus, any impairment on the capacity for synthesis, release, or re-uptake of catecholamines would result not only in lower concentrations of catecholamines in the active tissues, but as a diminished functional response as well. Evidence for such an impaired catecholamine response in the older animals is suggested by diminished cardiac catecholamine levels. While there was a trend for the tissue catecholamine levels to decline with age in the liver and kidney, the only significant difference
MAZE0
occurred between the 6- and 27-month-old animals in the kidney. This may be related to the degree of recruitment and distribution of blood to these tissues during exercise. It is evident that during the course of exercise, endurance training reduces the amount of circulating catecholamines in response to a given workload.*“** It has been suggested that this observation is a result of increased sensitivity to catecholamines by receptors. Winder et al*’ demonstrated in humans that the time course for this training-induced blood catecholamine response is approximately three weeks. Therefore, the ten-week training regimen employed in the present study was of sufficient duration to induce such a response. The finding in the present study that in response to strenuous exercise the catecholamine levels in the hearts of the young animals were greater in the untrained compared to the trained group provides further evidence for this hypersensitivity effect. However, in the 15 and 27-month-old animals the opposite response was observed in which the trained animals had higher levels of cardiac catecholamines than did the untrained group. This suggests that when compared to the young animals some other factor(s) has intervened, which has limited the older untrained animals’ ability for catecholamine synthesis, release, or re-uptake during the exercise stimulus and that training has offset this decline to a certain extent. Regardless of the training state, the 15 and 27-month-old animals demonstrated a significantly lower catecholamine response compared to the younger animals. Ten weeks of endurance training can significantly retard this age-related functional decline. The sympathetic nervous system, as well as the endocrine system, plays a major role in enabling the organism to adapt and maintain homeostasis in response to physical stress. The finding that the 27-monthold trained animals demonstrated significantly greater NE and E levels in the heart after strenuous exercise suggests that these animals are better adapted to cope with the demands of physical stress than their age-matched control group. While increased levels of cardiac catecholamines do not necessarily imply increased performance, the catecholamines have a pronounced effect on myocardial metabolism and function including enhanced glucose metabolism, increased rate, contractility, and cardiac output. Lakatta et al*’ has shown that the myocardial function, as measured by maximal rate of tension development (dT/dt), active tension, and contraction duration, to be greatly diminished in the aged heart in response to catecholamine administration. However, regardless of age, the inotropic response of the myocardium was greater with increasing catecholamine concentrations. This, in part, could explain the reductions in maximal stroke volume, cardiac output, heart rate, and functional capacity observed with aging. Further, Wyatt et al29 demonstrated that endurance training significantly improves the catecholamine-induced enhancement of myocardial contractile function and adenylate cyclase activity in the cat. Therefore, it would appear that the 27-monthtrained animals have a functional advantage in their ability to respond to the demands of intense physical exercise compared to their age-matched controls. The training stimulus improves the animals’ ability to respond to physical stress
ET AL
both in terms of the absolute catecholamine response and in the myocardium’s ability to respond to the catecholamines presented to it. This suggests that although the ageassociated functional decline may be related to the “aging process ” itself, other factors such as disuse and sedentary lifestyles can promote such changes. The reader should be aware of two factors that could possibly influence catecholamine levels of the present study. First, while all the animals ran at the same relative intensity, the running times to fatigue during the graded exercise test varied with age and training status. This could potentially affect the plasma catecholamine content. However, two lines of evidence suggest that this would not significantly alter our interpretation of the data. The six-month-old trained animals ran significantly longer than did the untrained animals of the same age (18.7 v 11.2 minutes), yet the trained animals demonstrated significantly lower NE and E levels in the heart. Further, this trend was reversed in the 15 and 27-month-old animals as the trained animals demonstrated higher catecholamine values when compared to their agematched controls. This reversal in response clearly indicates that some age-related factor(s) has intervened, which has effected the training response of catecholamine metabolism. In addition, while there were very small differences in run times to fatigue between the IS- and 27-month-old animals (Tr = 14.6 v 12.8 : Un = 7.4 v 6.3), there were significant differences in catecholamine content with age in response to exercise (Fig I). The method of death in the present study was decapitation. This represents a strong stimulus to the animal and is known to markedly elevate plasma catecholamines. This, in conjunction with the stimulus of strenuous exercise, could explain the relatively high levels of E observed in the heart. The combination of several factors including (1) a small amount of trapped blood remaining in the heart, (2) uptake of E into cardiac sympathetic terminals,3”32 and (3) increased synthesis of E by chromaffin cells within the heart33*34 most likely contribute to the relatively high E values observed in the heart after strenuous exercise. Regardless, in subsequent studies in our laboratory examining NE turnover in various tissues with age, an anesthetic was used to kill the animals. While cardiac catecholamine levels were slightly depressed compared to the results of the present study (most noticeable at rest but statistically insignificant), a similar response was observed as heart catecholamine content, as well as NE turnover rates, were significantly depressed with age. Based on previous literature regarding changes in the ability for catecholamine synthesis with advancing age, together with the results of the present study, it would suggest that the mechanism responsible for the diminished catecholamine levels in the aged rats during strenuous exercise is, in part, a result of a reduced capacity for catecholamine synthesis. However, data on tissue catecholamines alone is insufficient to provide conclusive evidence relating to sympathetic activity. Differences in catecholamine concentrations may reflect changes in the ability for synthesis and release, volume distribution, and clearance as determined by local metabolism and re-uptake into nerve terminals. While a
TISSUE
CATECHOLAMINE
RESPONSE
607
TO EXERCISE
number of recent reports have provided evidence demonstrating that tissue and circulating norepinephrine levels are a reliable index of underlying sympathetic activity,3C37 properly designed NE turnover studies need to be conducted to determine the exact mechanism responsible. However, the
results of the present study clearly indicate an age-related decline in cardiac catecholamine levels in response to strenuous exercise and that ten weeks of endurance training can significantly attenuate this response.
REFERENCES
1. Julius S, Amery A, Whitlock LS, et al: Influence of age on the hemodynamic response to exercise. Circulation 36:222-230, 1967 2. Lakatta EC: Age-related alterations in the cardiovascular responses to adrenergic mediated stress. Fed Proc 39:3173-3177, 1980 3. Shephard RJ: Cardiovascular limitations in the aged, in Smith EL, Serfass RC (eds): Exercise and Aging: The Scientific Basis. Hillsider, NJ, Enslow 1981, pp 19-29 4. Astrand I, Astrand PO, Rodahl K: Maximal heart rate during work in older men. J Appl Physiol 14:562-566, 1959 5. Alpert NR, Gale HH, Taylor N: The effect of age on contractile protein ATPase activity and the velocity of shortening, in: Kavelar K, Tanz RD, and Roberts J (eds): Factors Influencing Myocardial Contractility. Orlando, Fla, Academic, 1967, pp 127133 6. Capasso JM, Malhotra A, Remily RM, et al: Effects of age on mechanical and electrical performance of rat myocardium. Am J Physiol 245:H72-H81, 1983 7. Gerstenblith G, Lakatta EG, Weisfeldt ML: Age changes in myocardial function and exercise response. Prog Cardiovasc Dis 19:1-21, 1976 8. Guarnieri T, Filburn CR, Zitnik G, et al: Contractile and biochemical correlates of B-adrenergic stimulation of the aged heart. Am J Physiol239:H501-H508.1980 9. Heller LJ, Whitehorn WV: Age-associated alterations in myocardial contractile properties. Am J Physiol222:1613-1619, 1972 10. Starnes JW, Beyer RE, Edington DW: Myocardial adaptations to endurance exercise in aged rats. Am J Physiol 245:560-566, 1983 1.1. Weisfeldt ML, Wright JR, Shreiner DP, et al: Coronary flow and oxygen extraction in the perfused heart of senescent male rats. J Appl Physiol 30:4449, 197 1 12. Conway J, Wheeler R, Sannerstedt R: Sympathetic nervous activity during exercise in relation to age. Cardiovasc Res 5:577581, 1971 13. Frolkis VV, Bezrukov VV, Bogatskaya LN, et al: NS Catecholamines in the metabolism and functions regulation in aging. Gerontologia 16: 129-l 40, 1970 14. Timiras PS: Developmental Physiology and Aging. New York, Macmillan, 1972, pp 542-563 15. Gey KF, Burkard WP, Pletscher A: Variation of the norepinephrine metabolism of the rat heart with age. Gerontologia 1 l:l11, 1965 16. Roberts J, Goldberg PB: Changes in basic cardiovascular activities during the lifetime of the rat. Exp Aging Res 2:487-517, 1976 17. Mazzeo RS, Brooks GA, Horvath SM: Effects of age on the metabolic responses to endurance training in rats. J Appl Physiol 57:1369-1374,1984 18. Hallman H, Farnebo L, Hamberger B, et al: A sensitive method for the determination of plasma catecholamines using liquid chromatography with electrical detection. Life Sci 23:1049-1052, 1978
19. Anton AH, Sayre DF: A study of the factors affecting the aluminum oxide-trihydroxyindole procedure for the analysis of catecholamines. J Pharmacol Exp Ther 138:360-375, 1962 20. Galbo H, Richter EA, Hoist JJ: Diminished hormonal responses to exercise in trained rats. J Appl Physiol 43:953-958, 1977 21. Winder WW, Hickson RC, Hagberg JM, et al: Traininginduced changes in hormonal and metabolic responses to submaximal exercise. J Appl Physiol46:766-77 1, 1979 22. Winder WW, Beattie MA, Holman RT: Endurance training attenuates stress hormone responses to exercise in fasted rats. Am J Physiol243:R179-R184,1982 23. De Schryver C, Strythagen J, Becsei I, et al: Effect of training on heart and skeletal muscle catecholamine concentration in rats. Am J Physiol217:1589-1592, 1969 24. Gordon R, Spector S, Sjoerdsma A, et al: Increased synthesis of norepinephrine and epinephrine in the intact rat during exercise and exposure to cold. J Pharmacol Exp Ther 153:440-447,1966 25. Nikkila EA, Torsti P, Penttila 0: Effects of fasting, exercise and reserpine on catecholamine content and lipoprotein lipase activity of rat heart and adipose tissues. Life Sci 4:27-35, 1965 26. Leon AS, Horst WD, Spit? N, et al: Heart norepinephrine levels after exercise training in the rat. Chest 67:341-343, 1975 27. Winder WW, Hagberg JM, Hickson RC, et al: Time course of sympathoadrenal adaptation to endurance exercise training in man. J Appl Physiol45:37&374, 1978 28. Lakatta EG, Gerstenblith GS, Angel1 CS, et al: Diminished inotropic response of aged myocardium to catecholamines. Circ Res 361262-269, 1975 29. Wyatt HL, Chuck L, Rabinowitz B, et al: Enhanced cardiac response to catecholamines in physically trained cats. Am J Physiol 234:H608-H613,1978 30. Rose11S, Axelrod J, Kopin IJ: Release of tritated epinephrine following sympathetic nerve stimulation. Nature 201:301, 1964 3 1. Stromblad BCR, Nickerson M: Accumulation of epinephrine and norepinephrine by some rat tissues. J Pharmacol Exp Ther 134:154-159, 1961 32. Iversen LL: The uptake of catechol amines at high perfusion concentrations in the rat isolated heart: A novel catechol amine uptake process. Br J Pharmacol25:18-33, 1965 33. Glassman PM, Angelakos ET, McNary WF: Catecholaminecontaining fractions of dog heart homogenates. Life Sci 4:17271734, 1965 34. Potter LT, Axelrod J: Subcellular localization of catecholamines in tissues of the rat. J Pharmacol Exp Ther 142:291-298, 1963 35. Christensen NJ: Catecholamines and sympathetic nervous activity in the elderly. Acta Med Stand 676:52-63, 1983 (suppl) 36. Rowe JW, Troen BR: Sympathetic nervous system and aging in man. Endocr Rev 1:167-179, 1980 37. Wallin BG, Sundolf G, Eriksson B, et al: Plasma noradrenaline correlates to sympathetic muscle nerve activity in normotensive man. Acta Physiol Stand 111:69-73, 1981